Method and apparatus for molecular analysis in small sample volumes

ABSTRACT

The interrogation of extremely small sample volumes can be accomplished with the present invention. Provided are probes having disposed thereon a plurality of domains forming an array, which is suitably a nanoarray. Also provided are methods of detecting molecules and molecular interaction events, retrieving and analyzing analytes, and delivering substances to cells or tissues using probes of the invention.

Understanding the nature of interactions between biomolecular andmolecular species at both cellular and sub-cellular levels is key to theinvestigation of strategies for treating disease. One emergingmethodology for elucidating the nature of molecular interactionsinvolves the use of microarrays. Microarrays are spatially organizeddomains of various molecular species, and are typically constructed onsolid supports arranged to facilitate rapid detection andcharacterization of molecular interaction events. Such events includeinteractions between biomolecules, antibodies and antigens, enzymes andsubstrates, and receptors and ligands, as well as biochemical andinorganic molecular events.

One benefit of microarray technology is the ability to provide a largenumber of test sites in a relatively small area. The size of thedeposition domains, and in turn, the entire array, is of particularimportance in determining the limits of sample volumes that can betested.

There are four approaches for building conventional microarrays known inthe art. These methods include mechanical deposition, in situphotochemical synthesis, “ink jet” printing and electronically drivendeposition. Currently available mechanical deposition techniques producedomains of 25 to 100 microns in diameter or larger. In situphotochemical procedures allow for the construction of arrays ofmolecular species at spatial addresses in the 1-10 micron size range andlarger. So-called “ink jet” methods produce domains in the 100 micronrange. Electronic deposition can produce domains whose size is limitedby the method used to construct the deposition electrode(s). Typicallythis is in the many micron diameter size range. However, cellular andsub-cellular molecular events take place in volumes many times smallerthan the above-described available domain sizes. An apparatus andmethods for interrogating extremely small sample volumes, would permitdirect analyses of living cells in vivo or in situ.

Such arrays and methods would afford increased throughput and reduce thecosts associated with array production and utilization. The arrays andmethods would permit one to analyze extremely small sample volumeswithout requiring amplification of the material to be tested. A methodof analyzing molecular events in living cells or tissue in near realtime would also represent a substantial advance in the art. What istherefore described is a device and analytical platform for theevaluation of samples with volumes consistent with the contents of asingle cell or smaller that provides for near real-time analysis,increased throughput and reduced costs.

The present invention includes an apparatus for analyzing a samplecomprising a probe having a plurality of domains disposed thereon,wherein the domains form an array. Suitably, the array is a nanoarray.The domains suitably comprise biomolecules selected from the groupconsisting of drugs, chemical groups, lipids, DNA, RNA, proteins,peptide species, carbohydrates, and any combination of these entities.Optionally, nanosensors are operably connected to one or more of thedomains.

The probe suitably comprises a microcantilever. In some embodiments, theprobe is a dual element probe or a multielement probe. Some embodimentsof a probe of the invention comprise at least one microdisrupterdisposed on the probe. Optionally, at least one microdisrupter comprisesa tip or pointed member. The invention also encompasses probescomprising at least one hydrophobic region. Also described areembodiments wherein a suitable molecular detection device is operablyconnected to the probe. Suitable molecular detection devices includescanning tunneling microscopes, atomic force microscopes, massspectrometers, fluorescence microscopes, flow cytometers, Ramanspectrometers, Infra-red spectrometers, UV spectrometers, electronicsystems, electrochemical systems, optical systems, magnetic andelectromagnetic systems, and mass measuring systems.

Another aspect of the invention includes a method of detecting amolecular interaction event comprising contacting a sample with a probehaving a plurality of domains disposed in an array, providing anincubation period, washing unbound molecules from the domains anddetecting the molecular interaction event. Suitably, the samplecomprises at least one cell or at least one cell lysate.

Also described is a method of detecting one or more molecules in asample comprising contacting the sample with a probe having a pluralityof domains disposed thereon, wherein the domains form an array, andwherein the domains are operably connected to one or more sensors,including nanosensors; and detecting binding of one or more molecules toone or more of the domains.

The present invention also provides a method of analyzing one or moreanalytes in a cell comprising disrupting a cell with a microdisrupterdisposed on a probe, wherein the probe has a plurality of domainsdisposed thereon, and wherein the domains form a nanoarray; passing thenanoarray through the membrane of the cell such that the nanoarraycontacts intracellular space; and detecting the binding of one or moreanalytes to the nanoarray. Suitably, the method further comprisespassing the probe through the nuclear membrane such that the nanoarraycontacts the intranuclear space. Alternatively, the method can compriseinserting the probe into a cellular organelle. Cellular organellessuitable for analysis are those selected from the group consisting of agolgi complex, a mitochondria, a lysosome, an endoplasmic reticulum, alipid raft, a cytoskeletal system, and any other physically orchemically definable cellular or sub-cellular domain or system.

The invention also encompasses a method of retrieving at least oneanalyte from a sample comprising contacting the sample with a probehaving a plurality of domains disposed thereon, wherein the domains forman array; and retrieving at least one analyte from the moleculardomains.

Also provided is a method of delivering at least one substance to a cellcomprising reversibly attaching at least one substance to a probe havinga plurality of domains disposed thereon, wherein the domains form anarray; passing the probe through the membrane of the cell into theintracellular space; and releasing at least one substance into theintracellular space. Suitably, reversibly attaching at least onesubstance to a probe comprises contacting the substance to the domainssuch that a binding event occurs. Suitable substances include drugs,chemical groups, lipids, DNA, RNA, proteins, peptide species,carbohydrates and any combination of these entities. A suitable means ofreversibly attaching comprises tethering at least one substance to atleast one domain with a protease substrate. Additional methods include,but are not limited to, photolytic tethers, temperature sensitivetethers, ionically sensitive tethers, and chemically sensitive tethers.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic view of several embodiments of the inventionshowing mechanical micro4disrupter features.

FIG. 2 depicts use of an aqueous bridge with a probe of the inventionhaving a microdisrupter and hydrophilic and hydrophobic domains.

FIG. 3 depicts the use of the invention for direct interrogation ofintracellular contents.

FIG. 4 depicts experimental data showing protein arrays created onmicrofabricated atomic force microscope probe cantilevers. The arraysare rendered fluorescent by reaction with a fluorophore-coupled antibodythat is specific for the deposited protein. The inset is a brightfieldimage showing the deposited protein domains prior to fluorescentlabeling.

FIG. 5 depicts brightfield micrographs of a variety of microfabricatedprobes.

FIG. 6 depicts a brightfield micrograph of a two-component (two protein)array deposited on a probe of the type shown in FIG. 5.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The interrogation of extremely small sample volumes can be accomplishedwith the present invention. Provided are apparatuses including probesfor analyzing a sample with an array. Suitable methods for using theprobes of the invention are also provided.

Probes

As used herein, a “probe” refers to any suitable mechanical structureupon which an array can be composed and which can be used to interrogatea sample of small volume. Suitable probes include microfabricatedstructures. “Microfabricated structures” are millimeter, sub-millimeteror sub-micron scale structures and are generated by techniques known inthe art including, but not limited to, laser ablation,electrodeposition, physical and chemical vapor deposition,photolithography, wet chemical and dry etching, injection molding,electron beam lithography, and X-ray lithography. Other suitable probestructures for use in the present invention include biologicalmicrostructures such as eyelashes, cochlear hair cells, flagellum andactin filaments. Microcantilevers are also considered to be suitable foruse as probes in the present invention and can include any of theabove-described structures anchored at one or more ends or surfaces. Anyportion of the cantilever can be used as a suitable anchor point. Insome cases there may be multiple anchor points.

Optionally, a probe of the invention may include “microdisrupters,”which, as used herein, are features that are suitable for disrupting acell. Two mechanical embodiments of the microdisrupter feature areexemplified and depicted in FIG. 1. “Disruption” of a cell includes anysuitable technique by which the interior of a cell is accessed.“Disrupting” includes, but is not limited to, puncturing, penetrating,perturbing, oscillating, sonicating and lysing. Structures or featuressuitable for use as microdisrupters include tips or pointed members,serrated edges, pores, annulas, spheres or spherical members, enzymessuch as lipases or proteases capable of digesting all or a part of acell membrane, hypotonic or hypertonic compositions capable of alteringthe osmotic pressure of a cell and thermal or electromagnetic energydelivery devices including, but not limited to, photodiodes, lasers,electrical sources, temperature sources and radiowave sources. It shouldbe noted that a probe having no additional microdisrupter disposedthereon may itself be used to access the interior of a cell throughmicromanipulation or the delivery of energy, enzymes or compositions asdescribed above.

Probes of the invention are not limited to single element structures.For example, dual or multi-pronged probes are included within the scopeof the invention. Each element, or “tine,” of a multi-pronged probe caninclude an array. Arrays on adjacent prongs can be identical or can bedifferent, having domains of different species, or even different typesof molecules. For example, one prong can have an array of DNA speciesand an adjacent prong can have an array of peptide species. Some prongsmay not have an array disposed thereon. Additional prongs, if present,may serve the further function of disruption as described above, and mayalso include microdisrupters disposed thereon.

Probes of the invention may include anti-wicking features to preventcapillary action from drawing the sample away from the array. Suitablefeatures include hydrophobic domains and mechanical structures that arephysical barriers to wicking. Hydrophobic domains may be disposed on thesurface of the probe or may be an integral component of the probe.Hydrophobic domains may comprise any portion of the probe, but aresuitably constructed so as to facilitate maintained contact between thesample and the array. In this regard, hydrophobic domains may be used inconjunction with hydrophilic domains, which are most suitably disposedadjacent to, or as a substrate for, the array. Mechanical structuresthat are suitable for use in preventing wicking include O-ringstructures, micro-dikes, micro-walls, bumps, protrusions, holes,cavities, filters and temperature gradients.

Arrays

As used herein an “array” refers to a plurality of spatially arrangeddomains disposed in known locations, or “addresses” on a probe of theinvention. A “nanoarray” is an array in which each domain has a totalarea of about 100μ² (a diameter of about 5.6μ for round domains, or aside dimension of about 10μ for square domains), and preferably a totalarea of less than about one micron. A “domain” or a “molecular domain”or an “affinity domain” is a discrete region of immobilized speciesincluding, but not limited to, chemical species, biomolecular speciessuch as nucleic acids and peptides, and molecular and sub-molecularspecies. Specific non-limiting examples include antibodies, DNA, RNA,normally or abnormally expressed cellular proteins, pathogens andantigens derived therefrom, reactive organic and inorganic chemicalgroups and multi-component complexes. It should be noted that as usedherein, “peptide species” can include single amino acids, peptides,polypeptides and/or proteins.

Domains may further include nanosensors coupled to the immobilizedspecies. “Nanosensor,” as the term is used herein, refers to anyreporter system that enables direct detection of interaction events ormolecular activities occurring on the micron or smaller scale. Theconstruction of suitable nanosensors for use in the present inventionare described in copending application Ser. No. 09/974,755, entitled“Nanoscale Sensor” which is incorporated herein by reference in itsentirety. Briefly, nanosensors provide for the monitoring of nanoscaleevents by the detection of measurable changes in physical position,mass, electrical capacitance, conductivity or resistance, resonancefrequency, resonance amplitude, morphology, kinetic energy, localtemperature, oxidation/reduction (redox) state, structural integrity,bonding energy or other properties of the array species. Suitablestructures for use as nanoscale sensors include carbon nanotubes,fullerene structures, nanobars and nanowires.

Arrays can be constructed on probes by any suitable methodology. Onetechnique used in the construction of ultraminiturized arrays suitablefor use in the present invention is described in copending applicationSer. No. 09/929,865, entitled “Nanoscale Molecular Arrayer,”incorporated herein by reference in its entirety. This techniqueoperates via piezoelectric, mechanical, magnetic or other methods formanipulation of a probe to deposit and reproduce domains smaller thanabout 1 micrometer to as little as ten nanometers or less. Briefly, asuitable method for constructing arrays includes loading depositionmaterials on a deposition probe and transferring the materials to adeposition substrate using an apparatus having X, Y and Z controllersfor manipulation of the probe, a humidity controller, and a controlcomputer. Additional optional components of an apparatus suitable forconstructing arrays include a force feedback monitor and an opticalmicroscope.

The ultraminiaturized attributes of some probes of the invention allowthe construction of arrays with dimensions on the scale of a few micronsand with molecular arrays formed from at least 2 to about 250 moleculardomains of smaller than 1 micrometer down to as little as 10 nanometersor less each.

Molecular Detection Devices

As used herein, “molecular detection devices” include devices suitablefor reporting microscopic or submicroscopic events on a macroscopicscale. The ability to measure events that occur on minute scales andreport these events in the macroscopic world is of clear utility. Onedevice suitable for the direct detection of molecular interaction eventsoccurring at the micro- or nano-scale level is the scanning probemicroscope. One type of scanning probe microscope is the atomic forcemicroscope (“AFM”). In atomic force microscopy, the interactions betweena sharp, micron-scale probe and a sample are monitored and regulated asthe probe raster scans over the sample. Extremely fine control of themotion of the AFM probe is achieved using piezoelectric crystals. TheAFM is capable of about two nanometer (or less) lateral resolution andless than one Angstrom vertical resolution. It can be operated in avacuum, in atmospheres of varying humidity or in physiological solution,and is capable of identifying and measuring molecular binding events innear-real time. The resolution of the AFM can be very high, even on theatomic scale in some cases.

In addition to its high spatial resolution, the AFM is capable ofexerting and detecting forces in the picoNewton (pN) range. This is theforce range relevant to the forces extant between and within molecules.Thus, the AFM can measure intermolecular, as well as intramolecularbonding, or “rupture,” forces. This is accomplished by repeated cyclingof the AFM probe through an approach/retract sequence. Moreover, the AFMcan measure a wide variety of other forces and phenomena, such asmagnetic fields, thermal gradients and viscoelasticity.

Ultraminiaturization of molecular arrays is the next step in theevolution of microarray methodologies. Through ultraminiaturization,vast increases in throughput can be achieved, along with reductions incosts. Moreover, ultraminiaturization allows for the utilization of suchsmall sample volumes that the methods necessary for recovery of samplematerials can be virtually non-invasive, thereby greatly enhancing thecomfort level of the sample donor. For example, rather than a painfultissue biopsy, a few cells obtained by a simple swab technique canprovide the same level of information. Ultraminiaturization of arrayswould allow for in situ, and even in vivo, detection of molecular andbiomolecular events in real time, without the need for sample retrieval.Nonetheless, to date, no viable methodologies or devices foraccomplishing these goals have been described.

Microscopic or submicroscopic events include intermolecular andintramolecular interaction events. One measurable intramolecular eventis known as a “rupture event,” and is defined herein as the forcenecessary to induce the breaking of intramolecular bonds. Other typicalevents that are suitably measured and reported by molecular detectiondevices include the binding of one molecular species to anothermolecular species via covalent, non-covalent, hydrophobic, electrostaticor hydrogen bonding, or a combination of these or other bondingmechanisms. Non-limiting examples useful in the investigation of diseaseand therapeutic strategies include antibody-antigen interactions,receptor-ligand interactions and enzyme-substrate interactions.

Methods of molecular detection suitable for use in the present inventioninclude inverse cyclic voltametry and other methods using electronicplatforms, including but not limited to piezoelectric, capacitance,electromagnetic and laser-based devices. Other methods include the useof chemical reactions, changes in mass, bonding force, redox state,structural integrity, fluorescence, absorbance, quenching, localstructural variation, kinetic energy, thermal energy, magnetic orelectromagnetic reactivity, radio energy generation or absorption,general energy state and radioactivity to report binding events.

As discussed, the atomic force microscope is one instrument that isparticularly useful in practicing an embodiment of the presentinvention. Other suitable instruments include scanning tunnelingmicroscopes, mass spectrometers, fluorescence microscopes, flowcytometers, Raman spectrometers, Infra-red spectrometers, UVspectrometers, electronic systems, electrochemical systems, opticalsystems, magnetic and electromagnetic systems, and mass measuringsystems. As discussed above, nanosensors can also be used to reportmolecular events. Coupling nanosensors to an electronic measuring deviceincluding, but not limited to an amp meter, conductivity meter,ohm-meter, or oscilloscope allows for the macroscopic detection ofbinding and other molecular events.

Molecular detection devices can be operably connected to probes of theinvention. As used herein, “operably connected” refers to electric,magnetic, mechanical, optical, pneumatic or other means of connectingthe probe and the molecular detection device such that the macroscopicreporting of the molecular interaction event can be made simultaneouslyor in near-real time.

Methods

Probes of the invention may be used in situ or ex situ. As used herein,“in situ” usage refers to direct detection or measurement of molecularor sub-molecular events upon introduction of the array at the site ofinterest. For example, in situ usage includes in vivo interrogation of asample with a probe. In contrast, “ex situ” usage refers to removing thesample from the site of interest prior to interrogation with the probe.

A probe of the invention can be used to directly interrogate a singleliving or non-living cell, as shown in FIG. 3. Methods of isolatingsingle living cells are known in the art. For example, U.S. Pat. No.6,420,105, incorporated herein by reference, describes a method ofisolating and harvesting a single cell from its organ tissue using adevice capable of collecting cells so that they remain substantiallyintact. Positioning and motion of the probe is accomplished usingpiezoelectric or similar motion control devices. In some embodiments, itis possible to specifically target subcellular domains such as thenucleus, or a specific organelle, such as a Golgi body. Suitably, aprobe having a pointed member or other microdisrupter device situatedthereon is inserted directly into a cell or positioned adjacent to thecell. Alternatively, a probe without a microdisrupter device can enter acell or the cell can be lysed by any suitable means prior tointerrogation. The components of the cellular environment are thenallowed to interact with the molecular array on the probe.

In some cases, the amount of applied vertical force exerted by the probeon the sample is regulated by monitoring the degree of flexion of theprobe using such methods including but not limited to strain gauges,optical lever systems, integrated piezo resistive methods, or othersuitable methods. Motion of the probe can be in the X, Y plane and inthe Z plane. In addition, ultrasonic energy can be imparted by rapidoscillations of the probe. These motions are accomplished using piezoceramic motion control mechanisms, mechanical methods or other methodsthat are known to skilled practitioners in the art.

The array can contact the sample by any suitable direct or indirectmeans. An example of an indirect means of contacting a sample includesthe use of an aqueous bridge, as shown and depicted in FIG. 2.Hydrophilic and hydrophobic domains on the probe can be advantageouslyused to maintain contact between the sample and the array. When anaqueous bridge is used, a small drop of fluid deposited on the samplecell is captured between the probe and the cell and supporting substrateas shown in FIG. 2. The sample is mechanically disrupted by motion ofthe probe and contact with the microdisrupter. As the sample isdisrupted, the materials released diffuse through the aqueous bridge andcontact the molecular domains on the probe. Specific capture agents onthe array bind to components of interest contained in the sample. Asdiscussed above, binding events are monitored by a variety of methodsincluding, but not limited to, atomic force microscopy, fluorescence,Raman and IR scattering, mass spectrometry, electronic signatures, orchanges in mechanical or resonance properties of the probe itself.

Biomarkers are one type of suitable target molecule for probes of theinvention. As used herein, a “biomarker” is any molecule that can beused as an identifier of a particular cell, cell type, cell state,physiological state of an organ, organ system, or whole organism,tissue, tissue type, tissue state, predisposition to disease includingbut not limited to cancer, drug tolerance, cytotoxicity effects andmental or psychological function. Typically, biomarkers are proteins,but can also be cell-surface peptides, intracellular peptides, lipids,carbohydrate moieties, RNA transcripts and/or DNA molecules, chemicalgroups, and/or circulating antigens.

Another suitable target for molecular analysis using methods of theinvention is body fluid. A “body fluid” may be any liquid substanceextracted, excreted, or secreted from an organism or tissue of anorganism. Body fluid may or may not contain cells. Body fluids ofrelevance to the present invention include, but are not limited to,whole blood, plasma, serum, urine, cerebral spinal fluid, tears,sinovial fluid, semen, mucus and amniotic fluid.

Probes of the invention can also be used to retrieve an analyte from acomplex solution. As used herein, an “analyte” refers to any substance,molecule of interest, biomolecule of interest, or particle for whichinformation is desired. In this aspect, the arrays of the inventionsuitably comprise molecular domains capable of reversibly binding theanalyte either for direct measurement on the probe, or for release andsubsequent measurement by further analytical techniques. As will beappreciated by those of skill in the art, this embodiment can also beused to concentrate an analyte in a complex solution.

A further embodiment of the present invention is a method of deliveringone or more substances to a living or non-living cell, tissue, ororganism. In this embodiment, the domains of an array are “loaded” withthe substance or substances to be delivered, which is suitably attachedto the molecular domains by, for example, a protease labile tetheringmolecule. Suitable protease labile tethering molecules comprise apeptide sequence that is susceptible to being hydrolyzed by one or moreproteases found in the target cell, tissue or organism. Somenon-limiting examples of protease labile tethering molecules includeshort-chain peptide substrates of serine proteases, metalloproteases,aspartate proteases and cysteine proteases. Additional tetheringmolecules include, but are not limited to, ion sensitive tethers (e.g.,leucine zippers, chelaters (EDTA)), temperature sensitive tethers (e.g.,PNA, DNA or RNA), photosensitive tethers, or chemically sensitivetethers. Substances that are suitable for delivery by probes of theinvention include genes, polynucleotides comprising coding sequences,enzymes involved in DNA replication, transcription or translation,enzymes involved in cellular metabolism or other processes, restrictionendonucleases, ligases, reactive species such as free radicals, drugcandidates and drugs. As will be appreciated, the molecular domains of aprobe can be loaded for delivery of multiple molecules of the samesubstance or different substances, which may or may not act in concert.For example, a gene of interest can be delivered to a living cellsimultaneously with enzymes that can be used to splice it into theappropriate site of the host DNA.

Additional details of the invention will become more apparent byreference to the following non-limiting examples.

EXAMPLES

Printing Proteins on Microcantilevers

Protein arrays with approximately 1 μm diameter spot sizes have beenprinted on microcantilevers using the method described herein. FIG. 4shows two examples of AFM cantilever onto which rabbit immunoglobulin G(IgG) has been printed. The IgG was placed in the pattern shown using ananoscale molecular arrayer (hereinafter, “NanoArrayer”) as described incopending application Ser. No. 09/929,865. Once printed, the IgG wasvisualized by forming a complex with a second antibody, anti-rabbit IgGantibody conjugated to the fluorescent reporter molecule Alexa-594 andobserved in a fluorescence microscope using the appropriate wavelengthfilters.

Gold-coated AFM cantilevers (dual leg design with various springconstants) were placed in the NanoArrayer. A deposition tool (amicrofabricated device for lacing molecules on surfaces) wasfront-loaded by immersion in a solution of the rabbit IgG containing 1mg/ml antibody in a solution lacking any non-volatile salt butcontaining glycerol and non-ionic detergent in distilled water. Thedeposition tool was removed from the loading solution and brought intocontact with the gold coated AFM probe under careful control of localhumidity and temperature (typical humidity >50% RH at RT) and thedeposition process accomplished by physical transfer of materials fromthe deposition tool to the gold surface. This process was repeated untilthe pattern shown in the figure was achieved. The gold-coatedmicrocantilever was then incubated in a blocking solution containingTris-HCl, pH 7.2, 100 mM casein for 30 minutes and rinsed briefly withdistilled water. The secondary antibody was then added in a bufferedsolution containing Tris-HCl, pH 7.2, 50 mM NaCl and incubated for 30minutes at room temperature. The cantilever was again briefly rinsedwith distilled water and viewed in a fluorescence microscope. Thepresence of fluorescence in discrete domains demonstrates that therabbit IgG was printed on the microcantilever as expected. Thisexperiment demonstrated that it is feasible to practice the inventionand print biomolecular patterns on microfabricated devices that are thesame size scale as a single cell.

Printing Proteins on Microfabricated Devices

In another embodiment, a protein pattern was printed on a speciallyconstructed microcantilever device (“probe”). The probe contained asharp point as the mechanical disrupter disclosed in this application.Examples of probes are shown in FIG. 5.

Solutions of rabbit and mouse IgG (Jackson ImmunoResearch, PA) werediluted to 1 mg/ml in phosphate buffered saline, pH 7.4 (PBS). Thesesolutions were mixed 1:1 with spotting buffer containing non-ionicdetergent, glycerol but no non-volatile salt, and approximately 0.5 μlof each was deposited onto a glass coverslip by hand pipetting andplaced on the NanoArrayer printing stage. These solutions then served asthe loading domains to front-load microfabricated depositions tools inthe NanoArrayer. The deposition tool was treated with UV light (254 nm)for 30 minutes to enhance the loading process by increasing thehydrophilicity of the tool. The deposition tool was loaded by immersionof its distal end in a spot of sample solution. The probe onto which anarray of antibodies were to be printed was immobilized on theNanoArrayer printing stage using double-stick tape. Arrays of the firstantibody solution were printed using time-controlled mechanical contactbetween the deposition tool and the surface of the probe. The depositiontool was washed in distilled water in between sample loads to ensure nocross-contamination of spotted antibodies. The deposition tool was thenreloaded with a second antibody and used to deposit a second array,adjacent to the first, on the probe. The results of this process areshown in FIG. 6, depicting two 3-spot arrays created on a single probeusing this method. Arraying was performed at room temperature with 55%relative humidity.

Post-Deposition Processing

Following printing the probe is incubated for 3 hours in the NanoArrayerenvironmental chamber at a relative humidity of 70% to allow completebinding of the deposited antibodies onto the probe surface. Afterhumidification the surface was be blocked by immersion in 100 mM casein(in distilled water) for 30 minutes at room temperature, followed by 3washes in PBS containing 0.2% Tween-20 (PBST).

Optical-Detection and Data Collection

Antibody spot morphology was assessed by tagging the depositedantibodies with Alexa-594 anti-mouse and Alexa-488 anti-rabbitantibodies (Molecular Probes). The arrays of antibodies on the probewere read on a standard fluorescence microscope (Nikon TE-2000 invertedmicroscope and with a Hamamatsu ORCA-ER 1.3 megapixel cooled CCDcamera). Data from an entire nanoarray experiment was captured in asingle image using a 40× or 60× objective. Data was analyzed using MediaCybernetics Array-Pro v. 4.5 software that was specifically developedfor microarray analysis and is suitable for nanoarray analysis as well.

Surface Chemistry for Tethering Biomolecules

For successful deposition of antibodies or other biomaterials on probes,the surface chemistry must supply uniform monolayer immobilization,maintenance of native antibody state with accessibility to moleculartargets in the sample, array stability, and negligible backgroundbinding. In the development of standard nanoarrays, multiple surfacechemistries have been tested. One non-limiting chemistry approach fordeposition onto the probes described in the above examples is an aminereactive self-assembled monolayer (SAM) on a gold coated probe. The SAMconsisted of a succinimide-terminated alkanethiolate that was specificfor primary amines on the molecule to be tethered. These surfacesexhibit high protein binding while active, but are easily deactivated byhumidification to yield a surface with very low non-specific bindingcharacteristics.

The probe was cleaned in water and ethanol, followed by treatment for 45minutes with UV and ozone (broad wavelength Hg-vapor bulb that createslocal ozone via reaction with oxygen). The probes were coated with 5 nmchromium followed by 10 nm of gold in an ion beam sputter. Immediatelyfollowing sputtering, probes were immersed in a 0.5 mM solution of DSU(dithiobis-succinimidyl undecanoate, DSU; Dojindo MolecularTechnologies, Inc., MD) dissolved in 1,4 dioxane, and incubated for 3hours at room temperature, Probes were washed and briefly sonicated in1,4 dioxane, blown dry, and stored at room temperature under dry N₂ gas.

A related approach is to use a compound having an alkanethiolate with apolyethylene glycol spacer and an epoxide terminal group. Thealkanethiolate will form a tight monolayer on gold, the PEG spacer willresist non-specific protein adsorption while allowing rotational freedomfor capture molecules, and the promiscuous epoxide functional group willreact with primary amines on the deposited proteins. Unreacted epoxideswill hydrolyze in the presence of water to yield a diol that should haveexcellent non-specific adsorption properties. The increased rotationalfreedom of captured molecules that will be realized on this surface maypositively impact access of the sample molecules by detection antibodiesand will therefore be tested for reverse-phase applications.

Additional methods for tethering proteins and other biomoleculesinclude, but are not limited to, spontaneous adsorption, hydrophobicitymediated adsorption, covalent coupling sulfur-gold binding, use of apolyethylene glycol spacer with various distal chemistries, silanemediated covalent coupling, ionic binding, electrostatic binding andbiomolecular binding to pre-existing molecular layers includingprotein-protein, protein-nucleic acid, receptor-ligand and nucleicacid-nucleic acid interactions.

The following prophetic examples describe uses of the devices createdusing the invention.

Prenatal and Neonatal Screening

A small amount of prenatal (e.g., amniotic) or neonatal material isobtained. This material may be a blood sample, serum sample, body fluid,cell sample or any other biological sample for which a genetic orbiomarker screen is desired. In the case of blood, a microdrop of thematerial is prepared by pipetting onto a glass slide that is maintainedin a humid environment to prevent evaporation. A nanoarray probe isbrought into close proximity to the microdrop and inserted into the dropto allow the biomaterials on the drop to contact the molecular domainson the chip on a tip. After a suitable incubation period, the probe isremoved from the microdrop, and the array is washed and analyzed byfluorescence, atomic force microscopy, or other methods known to thosepracticing the art.

In an alternative embodiment of this example, a small number of cellsare obtained from a subject and maintained in a living state on asuitable substrate such as a glass slide or silicon chip. A nanoarrayprobe is carefully introduced into a cell through the cell membrane andallowed to interact with materials within the cell's cytoplasm,nucleoplasm or other sub-cellular location. After a suitable incubationperiod, the probe is removed from the cell, rinsed and evaluated asdescribed above.

Forensics

Typical forensic samples include cellular materials, body fluids andtrace chemicals. In one application of the present invention, a bloodsample is recovered from a crime scene. There is insufficient materialto complete a protein-based biomarker screen or a DNA fingerprintanalysis without amplification. In one embodiment, a protein biomarkerarray on a probe of the invention is brought into proximity with thesample which has been resuspended in a minimal volume (less than onemicroliter) to maintain the highest concentration possible of low copynumber protein biomarkers. After a suitable incubation period, the probeis processed as described above and a protein biomarker profile isobtained and can be used as a “signature” to identify or rule out asuspect.

Minimally Invasive Cancer Diagnostics

In many cases, the acquisition of necessary biopsy material for adiagnostic cancer screen is a very painful process for the patient. Thisis largely due to the relatively large amount of biopsy materialnecessary for adequate testing. Use of an ultraminiturized array on amicroprobe greatly decreases the amount of required material for adiagnostic screen, opening the door to methodologies that enhancepatient comfort considerably. For example, rather than a major surgicalprocedure to obtain a suspect breast tumor, a relatively small needle isinserted into a tumor with minimal discomfort, and a small number ofsuspect cells is withdrawn. A cancer biomarker specific probe isjuxtaposed to the cells and either the insertion or disruption techniqueis used to analyze the cellular content for cancer specific biomarkers.

It is envisioned that certain tissue suspected of being malignant (e.g.,throat tumors) could be sampled by swabbing to obtain a few cells thatcould be interrogated and analyzed as described above.

Delivery and Release of Biomaterials into Cells

In this example, rather using the probe to recover materials, a reverseprocedure is carried out. A probe is “loaded” with a variety ofmaterials, for example, DNA splicing enzymes, that are bound to specificsites on the array. The probe is then inserted into a specific cell orgroup of cells. By using a protease labile tether method, thebiomaterials are released within the cells and allowed to carry outtheir bioactivity in a very cell specific fashion. This multiplexeddelivery of materials to specific cells provides for the retention ofmaterials in an unreacted, “dormant” state on the probe until they areinserted into the cells and allowed to mix. This is particularlyapplicable in situations calling for site-specific modification of cellsis desirable, such as in gene therapy embodiments.

Transgenic Analysis

In this example, the goal is to evaluate small numbers of cells fortheir ability to grow into healthy transgenic animals. It is known thatat early divisional stages of embryogenesis, it is possible to removesingle cells without disrupting the growth of the embryo, assuming theembryo is otherwise normal and healthy. However, embryos that aremorphologically normal can carry aberrant genes or metabolic anomaliesthat will result in unhealthy or dead newborns. To avoid this, it isdesirable to carry out a biomarker profile of the embryo at an earlystage. In this scenario, the probe is diagnostic for a group ofbiomarkers that are indicative of normal cellular growth and function. Asingle cell is removed from the embryo at an early stage. The probe isinserted into or used to disrupt the cell and the cell contents allowedto interact with the affinity domains on the probe. The probe issubsequently processed and the biomarker screen used to make adetermination as to the health and utility of the embryo long before theexpense and technical difficulty of carrying a defective transgenicanimal to term are encountered.

Complex Biopsy Screening

A popular method for isolating different cell types from complex tissuesis known as Laser Cell Microdissection (“LCM”). In this method, a laseris used to cause adherence of specific cells to an adhesive backingwhich is then removed with the cells intact. These cells can beprocessed by conventional PCR methods to amplify DNA content, but thecell number is typically far too low to enable processing of proteinprofiles. A probe of the invention carrying the desired proteinprofiling affinity agents on the array can be used, either by insertionor disruption, to analyze the protein content of these dissected cells.

It should be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to a method of detecting “a biological event” includes amethod of detecting multiple biological events. It should also be notedthat the term “or” is generally employed in its sense including “and/or”unless the content clearly dictates otherwise.

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated by reference.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1. An apparatus for analyzing a sample comprising a probe having aplurality of domains disposed thereon, wherein the domains form anarray.
 2. The apparatus of claim 1, wherein the array is a nanoarray. 3.The apparatus of claim 1, wherein the domains comprise one or morebiomolecules selected from the group consisting of drugs, drugcandidates, chemical groups, lipids, DNA, RNA, proteins, peptidespecies, carbohydrates, and any combination thereof.
 4. The apparatus ofclaim 1, further comprising nanosensors operably connected to one ormore of the domains.
 5. The apparatus of claim 1, wherein the probecomprises a microcantilever.
 6. The apparatus of claim 1, wherein theprobe is a dual element probe.
 7. The apparatus of claim 1, wherein theprobe is a multielement probe.
 8. The apparatus of claim 1, wherein thesample comprises a volume of about 50 femtoliters to about 10microliters.
 9. The apparatus of claim 1, further comprising at leastone microdisrupter disposed on the probe.
 10. The apparatus of claim 9,wherein at least one microdisrupter comprises a tip or pointed member.11. The apparatus of claim 1, wherein the probe further comprises atleast one hydrophobic region.
 12. The apparatus of claim 1, furthercomprising a molecular detection device operably connected to the probe.13. The apparatus of claim 12, wherein the molecular detection device isa scanning tunneling microscope, atomic force microscope, massspectrometer, fluorescence microscope, flow cytometer, Ramanspectrometer, Infra-red spectrometer, UV spectrometer, electronicsystem, electrochemical system, optical system, magnetic andelectromagnetic system, or mass measuring system.
 14. A method ofdetecting a molecular interaction event comprising: contacting a samplewith a probe having a plurality of domains disposed in an array;providing an incubation period; washing unbound molecules from thedomains; and detecting the molecular interaction event.
 15. The methodof claim 14 wherein the sample comprises at least one cell.
 16. Themethod of claim 14 wherein the sample comprises at least one celllysate.
 17. A method of detecting one or more molecules in a samplecomprising: contacting the sample a probe having a plurality of domainsdisposed thereon, wherein the domains form an array, and wherein thedomains are operably connected to one or more nanosensors; and detectingbinding of one or more molecules to one or more of the domains.
 18. Amethod of analyzing one or more analytes in a cell comprising:disrupting a cell with a microdisrupter disposed on a probe, wherein theprobe has a plurality of domains disposed thereon, and wherein thedomains form a nanoarray; passing the nanoarray through the membrane ofthe cell such that the nanoarray contacts intracellular space; anddetecting the binding of one or more analytes to the nanoarray.
 19. Themethod of claim 18, further comprising passing the probe through thenuclear membrane such that the nanoarray contacts intranuclear space.20. The method of claim 18, further comprising inserting the probe intoa sub-cellular species.
 21. The method of claim 20 wherein thesub-cellular species is selected from the group consisting of a golgicomplex, a mitochondria, a lysosome, an endoplasmic reticulum, a lipidraft and a cytoskeletal system.
 22. A method of retrieving at least oneanalyte from a sample comprising: contacting the sample with a probehaving a plurality of domains disposed thereon, wherein the domains forman array; and retrieving at least one analyte from the moleculardomains.
 23. A method of delivering at least one substance to a cellcomprising: reversibly attaching at least one substance to a probehaving a plurality of domains disposed thereon, wherein the domains forman array; passing the probe through the membrane of the cell into theintracellular space; and releasing at least one substance into theintracellular space.
 24. The method of claim 23 wherein reversiblyattaching at least one substance to a probe comprises contacting thesubstance to the domains such that a binding event occurs.
 25. Themethod of claim 23 wherein at least one substance is DNA, RNA, a peptidespecies, a chemical, a drug or a reactive species.
 26. The method ofclaim 23 wherein reversibly attaching comprises tethering at least onesubstance to at least one domain with a protease substrate, aphotolyzable tether, a chemically reactive tether, an ionically reactivetether or a thermally sensitive tether.
 27. A method of detecting an insitu molecular interaction event comprising: contacting a sample with aprobe having a plurality of domains disposed in an array; providing anincubation period; and detecting the molecular interaction event.